Clinically effective gene therapy protocols for various diseases would ideally utilize procedures for efficient and specific targeting of therapeutic genes to affected cells while maintaining stable transduction and long term expression. This would be accomplished by direct injection into the bloodstream followed by homing of the vector to the desired target cells or organs. Thus, there have been many attempts to develop targeted gene transduction systems based upon various viral vectors. Adenovirus and adeno-associated virus vectors have been used in targeted gene delivery strategies because of their simple binding and entry mechanisms. (Nicklin and Baker, Curr Gene Ther. 2, 273-293 (2002)) Although these vectors have been used successfully in vitro, for targeting to specific cells, their usefulness in vivo has been limited by their natural tropism (Muller et al., Nat. Blotechnol. 21, 1040-1046 (2003)), especially to liver cells (Martin et al., Mol. Ther. 8, 485-494 (2003)).
Oncoretroviral- and lentiviral-based vectors have several properties that make them ideal for use in gene therapy (Sandrin et al., Curr. Top. Microbiol. Immunol. 281:137-78, 137-178 (2003)). Efficient integration of retroviral DNA into the host genome enables stable long-term transgene expression. Unlike oncoretroviral vectors, lentiviral vectors are capable of transducing non-dividing cells. The application of specific targeting with retroviral vectors has been problematic and the few studies of retroviral vector targeting in living animals are not efficient (Martin et al., Mol. Ther. 5, 269-274 (2002); Jiang and Dornburg, Gene Ther. 6, 1982-1987 (1999)). Inserting ligands, peptides or single chain antibodies into the retroviral receptor binding envelope subunit has been the most common approach used to alter and/or restrict the host range of retroviral vectors (Han et al., Proc Natl. Acad Sci U.S.A. 92, 9747-9751 (1995); Jiang et al., J. Virol. 72, 10148-10156 (1998); Marin et al., J. Virol. 70, 2957-2962 (1996); Martin et al., J. Virol. 73, 6923-6929 (1999); Nilson et al., Gene Ther. 3, 280-286 (1996); Somia et al., Proc Natl. Acad Sci U.S.A. 92, 7570-7574 (1995); Valsesia-Wittmann et al., J. Virol 68, 4609-4619 (1994)). Another approach is bridging virus vector and cells by antibodies or ligands (Boerger et al., Proc Natl. Acad Sci U.S.A. 96, 9867-9872 (1999); Roux et al., Proc Natl. Acad Sci U.S.A. 86, 9079-9083 (1989)). In general, most strategies have suffered from inconsistent specificity and low viral titers as a result of modification of the retroviral envelope (Han et al., Proc Natl. Acad Sci U.S.A. 92, 9747-9751 (1995); Marin et al., J. Virol. 70, 2957-2962 (1996); Nilson et al., Gene Ther. 3, 280-286 (1996); Somia et al., Proc Natl. Acad Sci U.S.A. 92, 7570-7574 (1995); Valsesia-Wittmann et al., J. Virol 68, 4609-4619 (1994); Kasahara et al., Science 266, 1373-1376 (1994)). The modified envelope proteins appear to have specific binding activity but low fusion activity resulting in inefficient entry into cells (Martin et al., J. Virol. 73, 6923-6929 (1999); Zhao et al., Proc Natl. Acad Sci U.S.A. 96, 4005-4010 (1999)). In the absence of specific targeting, current strategies depend upon direct injection to a localized site (Akporiaye and Hersh, Curr. Opin. Mol. Ther. 1, 443-453 (1999)) or, as in the case of the only successful treatment of a heritable disease, X-linked SCID, ex vivo isolation, purification and transduction of target hematopoietic cells (Kohn et al., Nat. Med. 1, 1017-1023 (1995); Cavazzana-Calvo et al., Science 288, 669-672 (2000)).
Sindbis virus is a member of the Alphavirus genus (Schlesinger and Schlesinger, Fundamental Virology 523-539, Raven, Philadelphia (1996)). In mature Sindbis virions the plus-stranded RNA viral genome is complexed with the capsid protein to form an icosahedral nucleocapsid surrounded by a lipid bilayer embedded with two integral membrane glycoproteins, E1 and E2, that form a heterodimer and function as a unit. E1 and E2 are anchored in the membrane independently. E2 binds to the host cell receptor. E1 can mediate membrane fusion as long as it is exposed to the low pH of the endosome and in the absence of a specific interaction with a receptor. Monoclonal antibodies capable of neutralizing virus infection are usually E2 specific, and mutation of E2 is frequently associated with altered host range and virulence. E2 can be modified substantially yet retain viral infectivity. This property of E2 has been exploited to develop Sindbis virus vectors that target specific cells (Ohno et al., Nat Biotechnol. 15, 763-767 (1997)). However, since Sindbis virus vectors are cytotoxic (Tseng et al. Systemic tumor targeting and killing by Sindbis viral vectors, Nat. Biotechnol. (2003)) and unable to stably transduce their target cells, they cannot be used where stable expression is desired.
We previously found that the Sindbis virus envelope (with E1 and E2) is able to pseudotype oncoretroviruses and lentiviruses (Morizono et al., J. Virol., September; 75 (17):8015-20. 75, 8016-8020 (2001)). We used the flexibility of the Sindbis virus E2 protein to our advantage in developing oncoretroviral and lentiviral vectors with the capacity to target specific cells. We previously reported an oncoretroviral and lentiviral gene targeting system based on antibody-mediated specific binding of a modified Sindbis virus envelope (ZZ SINDBIS) that encoded the ZZ domain of protein A. We demonstrated that monoclonal antibodies directed to cell surface antigens can be used to redirect the target specificity of these vectors when pseudotyped with the modified Sindbis envelope. Of particular note, the vectors maintained high viral titers, which could be further increased by simple ultracentrifugation.
Sindbis virus has a broad natural host range. The high-affinity laminin receptor (Wang et al., J. Virol. 66, 4992-5001 (1992)) and heparin sulfate are among the known receptors (Klimstra et al., J. Virol. 72, 7357-7366 (1998)). Their wide distribution and highly conserved nature may be in part responsible for the residual non-specific tropism observed with the ZZ SINDBIS pseudotyped vector. Accordingly, there exists a need for targeted retroviral vectors with decreased binding of endogenous receptors. The present invention fulfills this and other needs.